Fibrinolytic enzymes may be divided into two general classes. The first class of enzymes can be characterized by the ability to directly digest fibrin, and includes trypsin and plasmin. The second class indirectly digests fibrin by activating plasminogen. The latter class, comprising the plasminogen activators (i.e. urokinase and tissue plasminogen activator (tPA)), can be further characterized based on immunological criteria, molecular weight and polypeptide composition (see Collen et al., Thromb Haemostas, 48:294-296 (1982)).
In general, the plasminogen activators tend to have a higher substrate specificity than trypsin or plasmin. For example, while both tPA and trypsin recognize an arginine or lysine at the scissile bond site of a substrate, e.g., --X--Y--Arg--.dwnarw.--Z--Z'--, trypsin is capable of hydrolyzing a much broader range of peptide sequences. In fact, trypsin is capable of autolysis. tPA by contrast appears to hydrolyze a specific peptide loop in plasminogen and does not autolyze, owing to its high specificity.
The purification of plasminogen activators, most notably tPA, has been the focus of extensive research in recent years. Various protocols have been described for the purification of tPA including: salt precipitation, e.g. ammonium sulfate precipitation (see Rijken et al., Biochem Biophys Acts, 580:140 (1979), Meyhack et al., EP-A-143,081); ion exchange chromatography, e.g., zinc chelate agarose (see, Rijken et al., supra, Collen et al., U.S. Pat. No. 4,752,603, Dodd et al., FEBS, 209(1):13 (1986)), SP-Sephadex.RTM. (see, Kruithof et al., J. Biochem, 226:631-636 (1985)), and CM-Sepharose.RTM. (see, Murakami et al., U.S. Pat. No. 4,552,760). Another commonly used technique in conjunction with the method(s) disclosed above is size exclusion chromatography as taught by Collen et al., U.S. Pat. No. 4,752,603, Murakami et al., U.S. Pat. No. 4,552,760, and Rijken et al., Biochem Biophys Acts, 580:140 (1979).
In addition, various affinity chromatography ligands have been used in the purification of tPA. For example, Wallen et al. (Eur J Biochem, 133:681-686 (1983)) disclose the use of an anti-porcine tPA affinity ligand. Meyhack et al. (EP-A-143,081) disclose anti-tPA antibodies and the Erythrina latissima trypsin inhibitor (referred to as ETI or DE-3). Dodd et al. (FEBS, 209(1):13 (1986)) report purification of tPA using lysine as an affinity ligand. Murakami et al. (U.S. Pat. No. 4,552,760) suggest using a fibrin Sepharose.RTM. column for tPA purification. Wilson et al., (EP-A-113,319) report several purification schemes, including aminobenzamidine Sepharose.RTM. and DE-3 Sepharose.RTM.; and Wei et al. (EP-A-178,105) disclose the use of a dye (i.e. Trisacryl blue) as an affinity ligand.
Most of these methods are not appropriate for the large scale production of tPA, as they are inefficient in product recover or are only partially effective in removing impurities. Large scale purification methods which employ immunoaffinity chromatography (e.g., Wallen et al. (Eur J Biochem, 133:681-686 (1983)) and Meyhack et al. (EP-A-143,081)) are limited by the cost of the antibody resin, the difficulty in sterilizing this resin and by the potential for the antibodies, or fragments thereof, to leach into the recovered tPA.
Hence the need for a cost-effective affinity ligand to purify plasminogen activators remains. In order to obtain a high degree of purity, a ligand with a high avidity towards a plasminogen activator is needed. The problem then, is to identify such ligands with a high avidity for plasminogen activators, yet without such high avidity that the plasminogen activator cannot be desorbed without denaturation.